Multivalent and multispecific antigen-binding protein

ABSTRACT

A multivalent antigen-binding protein comprises a first polypeptide comprising, in series, three or more variable domains of an antibody heavy chain and a second polypeptide comprising, in series, three of more variable domains of an antibody light chain, said first and second polypeptides being linked by association of the respective heavy chain and light chain variable domains, each associated variable domain pair forming an antigen binding site. Methods for their production and uses thereof, in particular for therapeutic and diagnostic applications, are disclosed.

FIELD OF THE INVENTION

The present invention relates to multivalent and multispecific antigenbinding proteins, methods for their production and uses thereof. Inparticular, the invention relates to binding proteins comprisingpolypeptides which associate to form multivalent or multispecificmultimers.

BACKGROUND OF THE INVENTION

Antibodies are protein molecules having a structure based on a unitcomprising four polypeptides, two identical heavy chains and twoidentical light chains, which are covalently linked together bydisulphide bonds. Each of these chains is folded in discrete domains.The C-terminal regions of both heavy and light chains are conserved insequence and are called the constant regions, comprising one or moreso-called C-domains. The N-terminal regions of the heavy and lightchains, also known as V-domains, are variable in sequence and determinethe specificity of the antibody. The regions in the variable domains ofthe light and heavy chains (V_(L) and V_(H) respectively) responsiblefor antigen binding activity are known as the hypervariable orcomplementarity determining regions (CDR). Natural antibodies have atleast two identical antigen-binding sites defined by the association ofthe heavy and light chain variable regions.

It is known that proteolytic digestion of an antibody can lead to theproduction of antibody fragments. Such fragments, or portions, of thewhole antibody can exhibit antigen binding activity. An example of abinding fragment is an F_(ab) fragment which comprises a light chainassociated with the V_(H) and C_(H1) domains of a heavy chain. Thebivalent F(ab¹)₂ fragment comprises two such F_(ab) fragments connectedtogether via the hinge region, giving two antigen binding sites. F_(v)fragments, consisting only of the V-domains of the heavy and lightchains associated with each other may also be obtained. These F_(v)fragments are monovalent for antigen binding. Smaller fragments such asindividual V-domains (domain antibodies or dABs, Ward et al Nature, 341,544 (1989) and individual CDR's (Williams et al, Proc. Natl. Acad. Sci,U.S.A., 86, 5537 (1989)) have also been shown to retain the bindingcharacteristics of the parent antibody although generally most naturallyoccurring antibodies need both a V_(H) and V_(L) to retain fullimmunoreactivity.

Antibody fragments comprising V_(H) and V_(L) domains associatedtogether to have antigen binding activity have also been described. Thesingle chain F_(v) fragment (scFv) comprises a V_(H) domain linked to aV_(L) domain by a flexible polypeptide linker such that the domains canassociate to form an antigen binding site (see, for example,EP-B-0281604, Enzon Labs Inc).

Microbial expression systems for producing active antibody fragments areknown in the literature. The production of Fab in various hosts such asE. coli. (Better et al, Science, 240, 104, (1988)), yeast (Horwitz etal, Proc. Natl. Acad. Sci, U.S.4, 85, 8678 (1988)) and the filamentousfungus Trichoderma reesei (Nyyssönen et al, Bio/Technology, 11, 591(1993)) have previously been described, for example. It is also knownthat plants can be used as hosts for the production of SCFv fragments(Owen et al, Bio/Technology, 10, 790 (1992)) as well as wholeantibodies.

An advantage of using antibody fragments rather than whole antibodies indiagnosis and therapy lies in their smaller size. They are likely to beless immunogenic than whole antibodies and more able to penetratetissue. A disadvantage associated with the use of fragments such as theF_(ab), F_(v), and S_(c)F_(v) antibody fragments described above,however is that they have only one binding site for antigen binding ascompared to the two or more sites contained in the whole antibody,preventing polyvalent binding to the antigen and hence leading toreduced avidity.

In an attempt to overcome this problem, attention has been directed toproviding multivalent antigen binding proteins, that is binding proteinshaving more than one antigen binding site. In addition, there has beeninterest in producing antigen-binding proteins having multiplespecificities capable of binding to different antigenic determinants andcontaining antigen binding domains derived from different sources.Antigen-binding proteins having distinct binding specificities may beuseful, for example, in targeting effector cells to target cells byvirtue of the specific binding of the different binding domains. By wayof illustration, a bispecific antigen binding protein having specificityfor both tumour cells and cytotoxic drugs may be used to targetspecifically cytotoxic drug to tumour cell in an efficient manner. Byavoiding the need for chemical modification, adverse immune responsesmay be avoided.

Hitherto, the potential application of multivalent and multispecificantigen binding proteins have been hindered by the difficulties ingenerating and purifying such molecules.

Recombinant antigen-binding proteins having two binding sites may beprepared by methods such as chemical cross-linking of cysteine residues,either through cysteine residues introduced at the C-terminus of theV_(H) of an F_(V) (Cumber et al, J.Immunol., 149, 120 (1992)), throughthe hinge cysteine residues in F_(ab) to generate (Fab¹)₂ (Carter et al,Bio/Tech., 10, 163 (1992)) or at the C-terminus of the V_(L) of an scFv(Pack and Plückthun, Biochemistry, 31, 1579 (1992)). Alternatively, theproduction of bivalent and bispecific antibody fragments based on theinclusion of F_(ab) fragments of C-terminal peptide sequences whichpromote dimerisation has been described. (Kostelny et al, (1992)J.Immunol., 148, 1547).

Bivalent or bispecific antibody fragments comprising a binding complexcontaining two polypeptide chains, one comprising two heavy chainvariable domains (V_(H)) in series and the other comprising two lightchain variable domains (V_(L)) in series are described in our pendingEuropean Patent Application No. 95307332.7.

Multivalent and/or multispecific antibody fragments are described in WO94/09131 (Scotgen Limited). Specific binding proteins having two bindingregions, contained at least in part on first and second polypeptidechains which chains additionally incorporate associating domains capableof binding to each other causing the polypeptide chains to combine aredisclosed therein. It is disclosed that the first and second bindingregions preferably are antibody antigen-binding domains, for examplecomprising V_(H) and V_(L) regions contained in a Fab fragment or in asingle-chain Fv fragment, or may be derived from just one of the V_(H)or V_(L) regions of an antibody. The associating domains may suitably bederived from an antibody and may be inter alia antibody V_(H) and V_(L)regions. It is further disclosed that using a V_(H)/V_(L) domaincombination to achieve association leads to the creation of asupplementary Fv domain such that the antibody produced may betrivalent. Schematic representations of the arrangements suggested in WO94/09131 to produce trivalent fragments are shown in FIG. 1A. WO93/11161 (Enzon Inc) describes multivalent antigen-binding proteinscomprising two or more single-chain protein molecules, each single chainmolecule comprising first and second polypeptides each comprising thebinding portion of the variable region of an antibody heavy or lightchain with the polypeptides being linked together via a peptide linker.Hypothetical trimers and tetramers are discussed, comprising three orfour single-chain antigen binding proteins as appropriate. Schematicrepresentations of the trivalent arrangements suggested are shown inFIG. 1B.

WO 91/19739 (Celltech Limited) discloses multivalent antigen bindingproteins comprising an Fv fragment bound to at least one further Fvfragment by a connecting structure which links the Fv fragments togetherbut which maintains them spaced apart such that they can bind toadjacent antigenic determinants. Conveniently the connecting structureconsists of a spacing polypeptide and a linkage unit such as across-linking maleimide linker or a molecule which allows fornon-covalent binding. Particularly preferred connecting structures whichare disclosed are based on antibody joining and hinge region sequences.

SUMMARY OF THE INVENTION

According to the present invention there is provided a multivalentantigen binding protein comprising:

a first polypeptide comprising in series, three or more variable domainsof an antibody heavy chain; and

a second polypeptide comprising, in series, three or more variabledomains of an antibody light chain,

said first and second polypeptides being linked by association of therespective heavy chain and light chain variable domains, each associatedvariable domain pair forming an antigen binding site.

As used herein, the term multivalent means more than one antigen bindingsite.

Preferably the first polypeptide comprises three variable domains of anantibody heavy chain and the second polypeptide comprises three variabledomains of an antibody light chain, providing a trivalent protein.

It will be appreciated that the polypeptides may comprise heavy or lightchains, variable domains, as appropriate, or functional equivalentsthereof.

The respective heavy or light-chain variable domains may suitably belinked without any intervening linker. According to a preferredembodiment, however, the variable domains contained in the individualpolypeptides are linked by peptide linkers. Preferably the peptidelinker is flexible, allowing the variable domains to flex in relation toeach other such that they can bind to multiple antigenic determinantssimultaneously. It will be appreciated that the binding of the linker tothe individual heavy or light chain variable domains will be such thatit does not affect the binding capacity of the binding site formed bythe associated variable domain pair. Conveniently the peptide linkercomprises from 16 to 19 amino acid residues. A preferred, peptide linkerfor heavy chain domains is (Gly₄Ser)₃AlaGlySerAla (residues numbered121-139 of SEQ ID NO:27) and for the light chain domains is(Gly₄Ser)₃Val.

It will be appreciated that if two or more of the associated variabledomain pairs (V_(H)/V_(L) pairs) have the same antigen specificity, forexample if they are derived from the same parent antibody or fragmentthereof or from different antibodies which bind the same epitope, then abinding protein which binds more than one molecule of the same type willbe produced.

According to one embodiment, where the binding protein according to theinvention comprises three antigen binding sites which are able to binddifferent epitopes from each other, a trivalent trispecific protein isproduced.

In another embodiment, where the binding protein according to theinvention comprises three associated variable domain pair binding sites,two of which sites bind the same epitopes, a trivalent, bispecificprotein is provided. Where all three binding sites have the same antigenspecificity, a trivalent, monospecific binding protein is provided.

The invention also provides nucleotide sequences coding for thepolypeptides of the multivalent antigen binding protein according to theinvention and cloning and expression vectors containing such nucleotidesequences.

The invention further provides host cells transformed with vectorscontaining such nucleotide sequences and methods of producing suchpolypeptides by expression of the nucleotide sequences in such hosts.

The invention further provides a process for preparing a multivalentantigen binding protein as set forth above comprising:

(i) transforming one or more hosts by incorporating genes encoding saidfirst and second polypeptides;

(ii) expressing said genes in said host or hosts;

(iii) allowing said first and second polypeptides to combine to form theantigen binding protein.

Suitably the host or hosts may be selected from prokaryotic bacteria,such as Gram-negative bacteria, for example E. coli, and Gram-positivebacteria, for example B. subtilis or lactic acid bacteria, lowereukaryotes such as yeasts, for example belonging to the generaSaccharomyces Kluyveromyces or Trichoderma, moulds such as thosebelonging to the genera Aspergillus and Neurospora and highereukaroytes, such as plants, for example tobacco, and animal cells,examples of which are myeloma cells and CHO, COS cells and insect cells.A particularly preferred host for use in connection with the presentinvention is COS (monkey kidney) cells.

Techniques for synthesising genes, incorporating them into hosts andexpressing genes in hosts are well known in the art and the skilledperson would readily be able to put the invention into effect usingcommon general knowledge. Proteins according to the invention may berecovered and purified using conventional techniques such as affinitychromatography, ion exchange chromatography or gel filtrationchromatography.

The activity of the multivalent binding proteins according to theinvention may conveniently be measured by standard techniques known inthe art such as enzyme-linked immunosorbant assay (ELISA), radioimmuneassay (RIA) or by using biosensors.

The multivalent antigen binding proteins of the present invention maysuitably be used in diagnostics or therapy for example in targeting atumour cell with natural killer cells and cytotoxic agent. Other usesfor which the multivalent binding proteins according to the inventionare useful include those uses for which antibodies or fragments thereofare commonly used, including for immunoassays of a test sample and inpurification. According to a particular preferred embodiment,multi-enzyme complexes may be assembled, at a target, for example a cellsurface. As an illustration, multivalent binding proteins according tothe invention may be used to target cell killing enzymes such as anoxidase (for example glucose oxidase) and peroxidase (for examplehorseradish peroxidase) to a target species which is an antigeniccomponent of dental plaque, such as S. sanguis or S. mutans. Complexescomprising enzyme, coenzyme and target antigen may also conveniently beassembled.

Accordingly, the invention also provides compositions comprising themultivalent antigen binding proteins according to the invention,conveniently in combination with a cosmetically or pharmaceuticallyacceptable carrier, diluent or excipient. Methods of treatment using themultivalent antigen binding proteins according to the invention are alsoprovided.

For use in diagnosis or therapy, the multivalent antigen bindingproteins according to the invention may conveniently be attached to anappropriate diagnostically or therapeutically effective agent or carrierby methods conventional in the art.

An advantage of using multivalents antigen binding proteins according tothe invention over multivalent binding proteins prepared by existingtechniques known in the art is that the “self-assembling” association ofthe respective heavy and light chain variable domains to form themultivalent binding sites avoids the need for chemical coupling steps orthe introduction of linking residues to stabilise the multivalentconstructs, thereby minimising the risk of eliciting an immune responseto such molecules when the resulting multivalent binding proteins areused in therapy.

A particular advantage of molecules according to the present inventionis that they may conveniently be purified straight from the supernatantusing conventional purification techniques. As they are self-assembling,there is no need to purify individual subunits prior to coupling as inexisting techniques.

The present invention may be more fully understood with reference to thefollowing description, when read together with the accompanyingdrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show schematic representations of published arrangementsof heavy and light chain V-domain gene fragments that have beensuggested to produce trispecific or trivalent antibody fragments:

A) scFv1-VLa+scFv2-VHa (2 chains) WO 94/09131

B) Fab1-Vla+Fab2-VHa (4 chains) WO 94/09131

C) scFv1-VLa-CLa+scFv1-VHa-CHa (2 chains) WO 94/09131

D) Fab1-VLa-CLa+Fab2-VHa-CHa (4 chains) WO 94/09131

E) scFv1+scFv2+scFv3 (3 chains) WO 93/11161

F) VH1-VL2+VH2-VL3+VH3-VH1 (3 chains) WO 93/11161

FIGS. 2A1-2B2 show the nucleotide sequence of the EcoRI-HindIII insertof pGOSA.E2t containing DNA encoding pelB leader-VH4715-linker-VL3418and DNA encoding pelB leader-VL3418-linker-VH4715-hydrophil2 tag (SEQ IDNo. 1).

FIGS. 3A1-3A3 show the nucleotide sequence of the HindIII-EcoRI insertof plasmid scFv.Lys with DNA encoding pelB leader-VHLys-linker-VLLys(SEQ ID No. 2).

FIGS. 3B1-3B3 show the nucleotide sequence of the HindIII-EcoRI insertof plasmid scFv.4715.2t with DNA encoding pelB leader-VH4715.2t (SEQ IDNo. 3).

FIG. 4 shows the nucleotide sequence of the genomic leader sequence ofthe anti-NP antibody (Jones et al, Nature, 321, 522). Exon sequences areindicated with shaded boxes. NcoI and PstI restriction sites are in boldand underlined (SEQ ID No. 4).

FIG. 5 gives a schematic representation of the eukaryotic expressionvector pSV.51.

FIG. 6 gives an overview of the pUC19 double head (A) and triple head(B) constructs. The position of the oligonucleotides and the restrictionsites used for assembling double and triplehead pUC constructs areindicated.

FIG. 7

A) shows the origin of the VH-C-linker and VL-C-linker fragments.

B) gives a schematic representation of the construction of thepUC.19-triple-head vectors.

FIG. 8

A) gives a schematic representation of the construction of the Euka.VHand Euka.VL vectors.

B) gives a schematic representation of the construction of the pSV.VHexpression vectors.

C) gives a schematic representation of the construction of the pSV.VLexpression vectors.

FIG. 9 shows the expression of the trispecific Golysan proteins on anSDS-PAGE gel containing total COS culture supernatant. Crudesupernatants of COS cells transfected with pSV expression vectors wereseparated on SDS-PAGE gels. The proteins were transferred onto anitrocellulose membrane and the VH3 and VL3-2t were detected usinganti-VH and anti-hydrophil 2 tag specific monoclonal antibodiesrespectively. (A=anti-Hydro-II, B=anti-Hydro-II+anti-VH) Samples: M) LowMolecular Weight Markers, 1) pSV.K+pSV.V,2) pSV.K+pSV.W,3)pSV.M+pSV.V,4) pSV.M+pSV.W.

FIG. 10 shows the results of three ELISA's. Lysozyme, Glucose oxidaseand S. sanguis binding activity was determined in crude COS supernatantsby measuring 1) Lysozyme-Glucose oxidase (=LYSOX), 2) Glucose oxidase-S. sanguis (=GOSA) and 3) Lysozyme-S. sanguis (=LYSAN) bispecificbinding activities.

FIG. 11 shows the results of three ELISA's. Lysozyme, Glucose oxidaseand S. sanguis binding activity of purified Golysan.A (A) and Golysan.B(B) was determined by measuring 1) Lysozyme-Glucose oxidase (=LYSOX), 2)Glucose oxidase-S. sanguis (=GOSA) and 3) Lysozyme-S. sanguis (=LYSAN)bispecific binding activities.

FIGS. 12A and 12B show the nucleotide sequence of the EcoRI-HindIIIinsert of pUR.4124 containing DNA (see SEQ ID NO: 23) encodingV_(L)Lys-Linker-V_(H)Lys.

FIGS. 13A-13C show the nucleotide sequence of the HindIII-EcoRI insertof plasmid Fv.3418 (see SEQ ID NO: 24) containing DNA encoding pelBleader-V_(H)3418 and pelB leader-V_(L)3418.

FIGS. 14A-14C show the nucleotide sequence of the HindIII-EcoRI insertof plasmid Fv.4715-myc (see SEQ ID NO: 25) containing DNA encoding pelBleader-V_(H)4715 and pelB leader-V_(L)4715-Myc tag.

FIGS. 15A-15C show the nucleotide sequence of the HindIII-EcoRI insertof scFv.4715-myc containing DNA (see SEQ ID NO: 26) encoding pelBleader-V_(H)4715-Linker-V_(L)4715-Myc tag.

FIGS. 16A1-16A3 and 16B1-16B2 show the nucleotide sequence of theHindIII-EcoRI insert of pGOSA.E (see SEQ ID NO: 27) containing DNAencoding pelB leader-V_(H)4715-Linker-V_(L)3418 and pelBleader-V_(L)3418-Linker-V_(H)4715.

FIG. 16C gives an overview of the oligonucleotides and their positionsin pGOSA.E that can be used to replace V-domain gene fragments.

FIG. 17 shows the construction of plasmid pGOSA.A.

FIG. 18 shows the construction of plasmid pGOSA.B.

FIG. 19 shows the construction of plasmid pGOSA.C.

FIG. 20 shows the construction of plasmid pGOSA.D.

FIG. 21 shows the construction of plasmid pGOSA.E.

FIG. 22 shows the source of fragment PCR.I BstEII/SacI.

FIG. 23 shows the source of fragment PCR.IV XhoI/EcoRI.

FIG. 24 shows the source of fragment PCR.V SalI/EcoRI.

FIG. 25 shows the source of fragment PCR.III NheI/SacI.

FIG. 26 shows the source of fragment PCR.II SfiI/EcoRI.

Table 1 shows the nucleotide sequence of all oligonucleotides used inthe construction of the described double and triple head constructs.

Table 2 lists all pSV expression constructs described in thisspecification.

The following examples are provided by way of illustration only:

EXAMPLES General Experimental

Strains, Plasmids and Media

All cloning steps were performed in E. coli JM109 or E. coli XL-1 Blue.Cultures were grown in 2×TY/Amp/Glucose medium (16 g tryptone, 10 gyeast extract, 5 g NaCL per liter H₂O supplemented with 2% glucose and100 g/ml ampicillin). Transformations were plated out on SOBAG plates(20 g tryptone, 5 g yeast extract, 15 g agar, 0.5 g NaCl per liter H₂Oplus 10 mM MgCl₂, 2% glucose, 100 μg/ml ampicillin). The bicistronic E.coli vectors used are derivatives of pUC19. The COS expression vectorpSV.51 (LMBP strain nr 1829) was obtained from the LMBP Culturecollection (Laboratory of Molecular Biology University Gent). COS-1cells (ECACC No: 88031701;

African green monkey kidney cells) were obtained from the EuropeanCollection of Animal Cell Cultures (ECACC). All tissue culture reagentswere from Gibco BRL (Life Technologies, Paisley, UK)

DNA Manipulations

Oligonucleotides and PCR

The oligonucleotide primers used in the PCR reactions were synthesizedon an Applied Biosystems 381A DNA Synthesiser by the phosphoramiditemethod. The primary structures of the oligonucleotide primers used inthe construction of the trispecific pSV constructs (Table 2) are shownin Table 1. Reaction mixture used for amplification of DNA fragmentswere 10 mM Tris-HCl, pH8.3, 2.5 mM MgCl₂, 50 mM KCl, 0.01% gelatin(w/v), 0.1% Triton X-100, 400 mM of each dNTP, 5.0 units of Vent DNApolymerase (New England Biolabs), 100 ng of template DNA, and 500 ng ofeach primer (for 100 μl reactions). Reaction conditions were: 94° C. for4 minutes, followed by 33 cycles of each 1 minute at 94° C., 1 minute at55° C., and 1 minute 72° C.

Plasmid DNA\Vector\Insert Preparation and Ligation\Transformation

Plasmid DNA was prepared using the ‘Qiagen P-100 and P-500 Midi/Maxi-DNAPreparation’ system. Vectors and inserts were prepared by digestion of10 μg (for vector preparation) or 20 μg (for insert preparation) withthe specified restriction endonucleases under appropriate conditions(buffers and temperatures as specified by suppliers). Klenow fill-inreactions and dephosphorylation with Calf Intestine Phosphorylase wereperformed according to the manufacturers instructions. Vector DNA's andinserts were separated through agarose gel electrophoresis and purifiedwith DEAE-membranes NA45 (Schleicher & Schnell) as described by Maniatiset al. (Molecular cloning: a Laboratory manual, Cold Spring Harbour,N.Y. (1982)) Ligations were performed in 20 μl volumes containing 30 mMTris-HCl pH7.8, 10 mM MgCl₂, 10 mM DTT, 1 mM ATP, 300-400 ng vector DNA,100-200 ng insert DNA and 1 Weiss unit T₄ DNA ligase. After ligation for2-4 h at room temperature, CaCl₂ competent E. coli JM109 or XL-1 Blue(Maniatis et al) were transformed using 7.5 μl ligation reaction. Thetransformation mixtures were plated onto SOBAG plates and grownovernight at 37° C. Correct clones were identified by restrictionanalysis and verified by automated dideoxy sequencing (AppliedBiosystems).

Restriction Digestion of PCR Products

Following amplification each reaction was checked for the presence of aband of the appropriate size by agarose gel electrophoresis. One or two100 μl PCR reaction mixtures of each of the PCR reactions, togethercontaining approximately 2-4 μg DNA product were subjected tophenol-chloroform extraction, chloroform extraction and ethanolprecipitation. The DNA pellets were washed twice with 70% ethanol andallowed to dry. Next, the PCR products were digested overnight (18 h) in200 μL 1×Buffer with excess of the appropriate restriction enzyme.

Transformation of COS Cells

Cos-1 cells were maintained in DMEM culture medium with glutamine (2mM), Penicillin (100 U/mL), streptomycin (100 g/mL) containing 10%F.C.S. For transient transfection assays 1-3×10⁵ COS-1 cells were seededin 3 cm-diameter tissue culture dishes (2 mL). The cells were incubatedat 37° C. in a CO₂ incubator until cells were 50-80% confluent(overnight). For each transfection the following mixes were prepared: A)1 μg of each of the specified DNA's in 100 μL Opti-MEM-I Reduced SerumMedium, B) 1 μL LipofectAmine in 100 μL Opti-MEM-I Reduced Serum Medium.Mixes A and B were combined (gently). After allowing the DNA-liposomecomplexes to form for 30-45 minutes at room temperature, 0.8 mLOpti-MEM-I Reduced Serum Medium was added to each lipid DNA complexcontaining tube. The COS-1 cells were washed once with 2 mL ofOpti-MEM-I Reduced Serum Medium and overlayed with the diluted complexsolution. The COS-1 cells were incubated for 5 hr at 37° C. Followingincubation, 2 mL growth medium was added. 20 hours followingtransfection the medium was replaced with 2 mL fresh growth mediumcontaining 0.1 mM Na-butyrate. After 48 hours incubation at 37° C. thesupernatant was harvested and assayed for the presence of antibodyfragments.

ELISA

A) GOSA: Glucose Oxidase and S. sanguis Binding Activity

96 well ELISA plates (Greiner HC plates) were activated overnight at 37°C. with 200 μl/well of a 1/10 dilution of an overnight culture ofStreptococcus sanguis cells in 0.05 M sodium carbonate buffer pH9.5 wasused to sensitise each well. Following one wash with PBST, the antigensensitised plates were pre-blocked for 1 hour at 37° C. with 200 μ1/wellblocking buffer (1% BSA, 0.15% Tween in PBS). 50 μl COS culturesupernatants (neat or diluted with PBS) plus 50 μl blocking buffercontaining glucose oxidase (50 μg/ml) was added to the Streptococcussanguis sensitised plate and incubated for 2 hours at 37° C. Following 4washes with PBS-T, bound glucose oxidase was detected by adding 100 μlsubstrate to each well (70 mM Na-citrate, 320 mM Na-phosphate, 27 mg/mlglucose, 0.5 μg/ml HRP, 100 μg/ml TMB). The colour reaction was stoppedafter 1 hour by the addition of 35 μl 2M HCl and the A450 was measured.

B) LYSOX: Lysozyme and Glucose Oxidase Binding Activity

96 well ELISA plates (Greiner HC plates) were activated overnight at 37°C. with lysozyme (50 μg/mL in 0.05M sodium carbonate buffer pH9.5; 200μl/well). Following one wash with PBST, the antigen sensitised plateswere pre-blocked for 1 hour at 37° C. with 200 μl/well blocking buffer(1% BSA, 0.15% Tween in PBS). 50 μl COS culture supernatants (neat ordiluted with PBS) plus 50 μl blocking buffer containing glucose oxidase(50 μg/ml) was added to the Streptococcus sanguis sensitised plate andincubated for 2 hours at 37° C. Following 4 washes with PBS-T, boundglucose oxidase was detected by adding 100 μl substrate to each well (70mM Na-citrate, 320 mM Na-phosphate, 27 mg/ml glucose, 0.5 μg/ml HRP, 100μg/ml TMB). The colour reaction was stopped after 1 hour by the additionof 35 μl 2M HCl and the A450 was measured.

C) LYSAN: S. sanguis and Lysozyme Binding Activity

96 well ELISA plates (Greiner HC plates) were activated overnight at 37°C. with 200 μl/well of a 1/10 dilution of an overnight culture ofStreptococcus sanguis cells in 0.05M sodium carbonate buffer pH9.5 wasused to sensitise each well. Following one wash with PBST, the antigensensitised plates were pre-blocked for 1 hour at 37° C. with 200 μl/wellblocking buffer (1% BSA, 0.15% Tween in PBS). 50 μl COS culturesupernatants (neat or diluted with PBS) plus 50 μl blocking buffer wasadded to the Streptococcus sanguis sensitised plate and incubated for 2hours at 37° C. Following 4 washes with PBS-T, 50 μL blocking buffercontaining Alkaline-Phosphatase conjugated Lysozyme (100 μg/mL). UnboundLysozyme was removed by 4 washes with PBS-T. Bound Lysozyme was detectedby adding 100 μL substrate solution to each well (1 mg/ml pNPP in 1Mdiethanolamine, 1 mM MgCl₂). After 1 hour the A405 was measured.

EXAMPLE 1 Construction of the pSV.Golysan Expression Vectors

The construction of the pSV COS expression vectors consisted of threestages:

1A): Assembly of 2 heavy chain variable domains and 2 light chainvariable domains in a pUC based E. coli expression vector thusconstructing the VH_(A)-VH_(B)and VL_(A)-VL_(B) modules respectively.

1B): Assembly of 3 heavy chain variable domains and 3 light chainvariable domains in a pUC based E. coli expression vector thusconstructing the VH_(A)-VH_(B)-VH_(C) and VL_(A)-VL_(B)-VL_(C) modulesrespectively.

2) Linking the VH_(A)-VH_(B), VH_(A)-VH_(B)-VH_(C) and VL_(A)-VL_(B),VL_(A)-VL_(B)-VL_(C), to the genomic anti-NP leader sequence in theintermediate “EUKA” vectors to ensure efficient secretion by COS cells.

3) Inserting the leader-VH_(A)-VH_(B), leader-VH_(A)-VH_(B)-VH_(C) andleader-VL_(A)-VL_(B), leader-VL_(A)-VL_(B)-VL_(C) as XbaI/XbaI fragmentsdownstream of the SV40 promoter in the COS expression vector pSV.51.

ad.1) E. coli Expression Vectors

The E. coli expression vectors are derivatives of pUC.19 containing aHindIII-EcoRI fragment that in the case of the scFv.lys-myc contains apelB signal sequence fused to the 5′ end of the heavy chain V-domainthat is directly linked to the corresponding light chain V-domain of theantibody through a connecting sequence that codes for a flexible peptide(Gly₄Ser)₃ thus generating a single-chain molecule. In the ‘double head’expression vector both the heavy chain and the light chain V-domains ofthe antibody are preceded by a ribosome binding site and a pelB signalsequence in an artificial dicistronic operon under the control of asingle inducible promoter. Expression of these constructs is driven bythe inducible lacZ promoter. The nucleotide sequence of theHindIII-EcoRI inserts of the scFv.lys-myc, scFv.4715.2t and pGOSA.E2tconstructs used for the generation of the trispecific antibody fragmentsare listed in FIGS. 3 and 2 respectively.

ad.1A) Assembly of Bi-Specific Fragments or Double Heads

The construct pGOSA.E2t (FIGS. 2 and 6A) is derived from the E. coliexpression construct pGOSA.E. The construction of pGOSA.E has beendescribed in detail in preparation 1 below.

In contrast with pGOSA.E, pGOSA.E2t contains a peptide tag at theC-terminus of the Variable light chain. Using oligonucleotides DBL3 andDBL.4 the VL4715 gene fragment was amplified using scFv.4715.2t as atemplate. The SalI/BamHI VH4715.2t PCR fragment and the Hydrophil-2 tagcontaining BamHI/EcoRI fragment from scFv.4715.2t (FIG. 3B) were used toreplace the SalI/EcoRI VH4715 fragment in pGOSA.E thus producingpGOSA.E2t.

The vector pGOSA.E2t and the oligonucleotides in Table 1 have beendesigned to enable most specificities to be cloned into the pGOSA.E2tconstruct (FIG. 6A). The upstream V_(H) domain can be replaced by anyPstI-BstEII V_(H) gene fragment obtained with oligonucleotides PCR.51and PCR.89. The oligonucleotides DBL.1 and DBL.2 were designed tointroduce SfiI and NheI restriction sites in the V_(H) gene fragmentsthus allowing cloning of those V_(H) gene fragments into the SfiI-NheIsites as the downstream V_(H) domain. Using this approach the followingVH_(A)-VH_(B) combinations were constructed: VH4715-VH3416,VH4715-VHlys, VH3418-VHlys, VHlys-VH3418.

All V_(L) gene fragments obtained with oligonucleotides PCR.116 andPCR.90 can be cloned into the position of the 3418 V_(L) gene fragmentas a SacI-XhoI fragment. A complication here however is the presence ofan internal SacI site in the 3418 V_(H) gene fragment. OligonucleotidesDBL.3 and DBL.4 are designed to allow cloning of V_(L) gene fragmentsinto the position of the 4715 V_(L) gene fragment as a SalI-BamHIfragment. A complication here however is the presence of an internalBamHI site in the hydrophil-2-tag gene fragment (2t). Using thisapproach the following VL_(A)-VL_(B) combinations were constructed:VL3418-VL4715.2t, VLlys-VL4715.2t and VLlys-VL3418.2t.

ad.1B) Assembly of Tri-Specific Fragments or Triple Heads

Amplification of the VH-linker fragments using either scFv(VH-linker-VL) or bi-specific constructs (VH-linker-VH) as template withthe primer combination DBL.1/DBL.5 (FIG. 7A) yields one of the buildingblocks for the construction of the VH_(A)-VH_(B)-VH_(C) modules. TheVH-linker DBL.1/DBL.5 PCR fragment is digested with SfiI and insertedinto the SfiI site that is present between the linker sequence and thedownstream V_(H) domain in all bi-specific constructs (FIG. 7B) thusproducing a VH_(A)-VH_(B)-VH_(C) module. Using this approach thefollowing VH_(A)-VH_(B)-VH_(C) combinations were constructed for thisfiling: VH4715-VHlys-VH3418 and VHlys-VH4715-VH3418.

Using a bi-specific construct (VL-linker-VL) as the template in anamplification reaction with the primer combination DBL.3/DBL.6 (FIG. 7A)yields the VL-linker building block for the construction of theVL_(A)-VL_(B)-VL_(C) modules. The VL-linker DBL.3/DBL.6 PCR fragment isdigested with SalI and inserted into the SalI site that is presentbetween the linker sequence and the downstream VL domain in allbi-specific constructs (FIG. 7B) thus producing a VL_(A)-VL_(B)-VL_(C)module. Using this approach the following VL_(A)-VL_(B)-VL_(C)combinations were constructed: VLlys-VL4715-VL3418.2t andVL3418-VLlys-VL4715.2t.

A schematic representation of the final tri-specific constructs is shownin FIG. 6B.

ad.2) Linking the Variable Region Domains to the Leader Sequence

The HindIII/EcoRI polylinker of pUC19 was replaced with a syntheticEcoRI/HindIII ‘Euka’ polylinker. This was achieved by annealing andinserting the synthetic oligonucleotides Euka.1 and Euka.2 (Table 1)into EcoRI/HindIII digested pUC19 vector. The resulting Euka.pUC vectorcontains all restriction sites needed for the subcloning of the leadersequence and the VH and VL domains. The NcoI/PstI genomic anti-NP leadersequence fragment was cloned into the NcoI/PstI digested Euka.pUC vectoryielding the Euka.VH construct (FIG. 8A).

Oligonucleotides ML.1 and ML.2 (Table 1) were used in an amplificationreaction to introduce a SacI site at the 3′ end of the leader sequencethat allows the construction of leader-VL fusions. The NcoI/SacI leadersequence PCR fragment was inserted into NcoI/SacI digested Euka.pUCvector yielding the Euka.VL construct (FIG. 8A).

The VH_(A)-VH_(B)and VH_(A)-VH_(B)-VH_(C) modules were excised from thepUC expression vectors as PstI/NheI fragments and inserted intoPstI/NheI digested Euka.VH vector (FIG. 8B). Using this approach thefollowing leader-VH_(A)-VH_(B) and leader-VH_(A)-VH_(B)-VH_(C)combinations were constructed for this filing: Euka.B:leader-VH4715-VH3418, Euka.D: leader-VH4715-VHlys, Euka.G:leader-VH3418-VHlys, Euka.K: leader-VH4715-VHlys-VH3418 and Euka.M:leader-VHlys-VH4715-VH3418.

The VH_(A)-VH_(B) and VH_(A)-VH_(B)-VH_(C) modules were excised from thepUC expression vectors as EcoRI-Klenow/SacI fragments and inserted intoNotI-Klenow/SacI treated Euka.VL vector (FIG. 8C). Using this approachthe following leader-VL_(A)-VL_(B) and leader-VL_(A)-VL_(B)-VL_(C)combinations were constructed: Euka.N: leader-VL3418-VL4715.2t, Euka.P:leader-VLlys-VL4715.2t Euka.S: leader-VLlys-VL3418.2t, Euka.V:leader-VLlys-VL4715-VL3418.2t and Euka.W: leader-VL3418-VLlys-VL4715.2t.

ad.3) Subcloning of Leader-Variable Domain Fusions Into the pSV.51Expression Vector

All leader-VH_(A)-VH_(B), leader-VH_(A)-VH_(B)-VH_(C),leader-VH_(A)-VH_(B) and leader-VH_(A)-VH_(B)-VH_(C) combinations wereexcised from the ‘Euka’ vectors as XbaI/XbaI fragments and subcloneddownstream of the SV40 promoter in pSV.51 (FIG. 5) by insertion into theXbaI site (FIGS. 8B and 8C). After confirmation of the correctorientation of the inserts the pSV expression vectors were used totransfect COS-1 cells (see Example 2). The pSV expression vectors usedare listed in Table 2.

EXAMPLE 2 Bifunctional Binding Activity of Golysan Triple Heads

This example describes the production of three types of bispecificbinding activity by COS-1 cells transfected with expression plasmidsencoding the corresponding VH_(A)-VH_(B)-VH_(C) and VL_(A)-VL_(B)-VL_(C)genes fragments.

1. Production of Antibody Fragments by COS-1 Cells

Supernatants of COS-1 cells transfected with combinations ofpSV-VH_(A)-VH_(B)-VH_(C) and pSV-VH_(A)-VH_(B)-VH_(C) expressionplasmids were separated on 10% SDS-PAGE and transferred ontonitrocellulose. The resulting Western blots were screened with amonoclonal antibody recognising a peptide sequence in framework 4 of theVH domains (region encoded by PCR.89: conserved in all used VH domains,{in-house reagent}) and/or a monoclonal specific for the hydrophil-2tag. As shown in FIG. 9 all supernatants contained products with theexpected molecular weight of the VH_(A)-VH_(B)-VH_(C) andVL_(A)-VL_(B)-VL_(C) fragments, indicating that the COS cells weresuccessfully tranfected and were secreting the produced antibodyfragments into the culture medium at detectable levels.

2. Bifunctional Binding Activity

Supernatants of COS-1 cells transfected with single pSV expressionplasmids and combinations of pSV expression plasmids were tested for theproduction of bifunctional binding activity using ELISA format:

Supernatants of COS-1 cells transfected with the bispecific positivecontrols ‘LYSAN’ (pSV.D+pSV.P), ‘LYSOX’ (pSV.G+pSV.S) and ‘GOSA’(pSV.B+pSV.N) only produced LYSAN, LYSOX and GOSA bispecific activityrespectively (FIG. 10). No significant cross reactivity was detected.

Supernatants of COS-1 cells transfected with only one expression vectorencoding either one of the VH_(A)-VH_(B)-VH_(C) fragments (pSV.K andpSV.M) or one of the VL_(A)-VL_(B)-VL_(C) fragments (pSV.V and PSV.W)did not exhibit any bispecific binding activity, indicating that nobackground binding or a specific binding activity is produced.

All tested supernatants of COS-1 cells transfected with an expressionvector encoding one of the VH_(A)-VH_(B)-VH_(C) fragments (pSV.K andpSV.M) and an expression vector encoding one of the VL_(A)-VL_(B)-VL_(C)fragments (pSV.V and pSV.W) showed significant levels of all threebifunctional binding activities LYSOX, GOSA and LYSAN.

These results show that COS cells transfected with expression vectorsencoding VH_(A)-VH_(B)-VH_(C) and expression vectors encodingVL_(A)-VL_(B)-VL_(C) fragments produce and secrete molecules thatcontain three binding activities. In this example those three activitiesare: Glucose Oxidase binding, S. sanguis binding and Lysozyme binding.Furthermore, the results illustrated in FIG. 10 clearly show that atleast two of these binding activity are present in one self assemblingmolecular complex. In this example those combinations are: GOSA (GlucoseOxidase+S. sanguis), LYSOX (Lysozyme+Glucose Oxidase) and LYSAN(Lysozyme+S. sanguis).

EXAMPLE 3 Trifunctional Binding Activity of Golysan Triple Heads

This example describes experiments that show that the three types ofbispecific binding activity that are produced by COS-1 cells transfectedwith expression plasmids encoding the corresponding VH_(A)-VH_(B)-VH_(C)and VL_(A)-VL_(B)-VL_(C) genes fragments are present in one selfassembling molecular complex.

Golysan.A (VHlys-VH4715-VH3418+VLlys-VL4715-VL3418.2t) and Golysan.B(VHlys-VH4715-VH3418+VL3418-VLlys-VL4715.2t) was purified by affinitychromatography. 100 ml supernatant of COS-1 cells transfected withexpression plasmids pSV.M/pSV.V (Golysan.A) or pSV.M/pSV.W (Golysan.B)were loaded onto a Lysozyme-Sepharose column (CNBr-Sepharose, Pharmacia;column was prepared according to the manufacturer's instructions). Afterextensive washes with PBS the bound Golysan antibody fragments wereeluted in 0.1M glycine buffer at pH=2.2. The fractions were neutralisedwith Tris and tested for the presence of trispecific binding activity.

As shown in FIG. 11 no bispecific binding activity was detect in thecolumn fall-through. All three bispecific binding activities (GOSA,LYSOX and LYSAN) were extracted from the COS-1 supernatant by passingover the Lysozyme affinity matrix. After acid elution all threebispecific binding activities (GOSA, LYSOX and LYSAN) were recoveredfrom the column. Since both Golysan.A and B were affinity purified basedon the ability to bind to Lysozyme, the finding that these moleculesalso bind S. sanguis and Glucose Oxidase shows that all three bindingactivities are present in one self assembling molecular complex.

Preparation 1

Construction of the pGOSA.E Double Head Expression Vector

In the pGOSA expression vectors, the DNA fragments encoding both theV_(H) and V_(L) of the antibody are preceded by a ribosome binding siteand a DNA sequence encoding the pelB signal sequence in an artificialdicistronic operon under the control of a single inducible promoter.Expression of these constructs is driven by the inducible lacZ promoter.The nucleotide sequence of the HindIII-EcoRI inserts of the plasmidspUR.4124 (SEQ ID NO. 23), Fv.3418 (SEQ ID NO. 24), Fv.4715-myc (SEQ IDNO. 25) and scFv.4715-myc (SEQ ID NO. 26) constructs used for thegeneration of the bispecific antibody fragments are given in FIGS.12-15, respectively. Moreover, a culture of E. coli cells harbouringplasmid scFv.4715-myc and a culture of E. coli cells harbouring plasmidFv.3418 were deposited under the Budapest Treaty at the NationalCollection of Type Cultures (Central Public Health Laboratory) in London(United Kingdom) with deposition numbers NCTC 12916 and NCTC 12915,respectively.

In agreement with Rule 28 (4) EPC, or a similar arrangement for a Statenot being a Contracting State of the EPC, it is hereby requested that asample of such deposit, when requested, will be submitted to an expertonly.

The construction of pGOSA.E (see FIG. 16 for the HindIII-EcoRI insert ofpUC19) involved several cloning steps. The appropriate restriction sitesin the various domains were introduced by PCR directed mutagenesis usingthe oligonucleotides listed in Table 1 below.

The construction of pGOSA.E involved several cloning steps that produced4 intermediate constructs pGOSA.A to PGOSA.D (see FIGS. 17-21). Thefinal expression vector pGOSA.E and the oligonucleotides in Table 1 havebeen designed to enable most specificities to be cloned into the finalpGOSA.E construct (FIG. 16c). The upstream V_(H) domain can be replacedby any PstI-BstEII V_(H) gene fragment obtained with oligonucleotidesPCR.51 and PCR.89 (see Table 1). The oligonucleotides DBL.1 and DBL.2(see Table 1 ) were designed to introduce SfiI and NheI restrictionsites in the V_(H) gene fragments thus allowing cloning of those V_(H)gene fragments into the SfiI-NheI sites as the downstream V_(H) domain.All V_(L) gene fragments obtained with oligonucleotides PCR.116 andPCR.90 (see Table 1) can be cloned into the position of the VL.3418 genefragment as a SacI-XhoI fragment. A complication here however is thepresence of an internal SacI site in the V_(H).3418gene fragment.Oligonucleotides DBL.3 and DBL.9 (see Table 1) are designed to allowcloning of V_(L) gene fragments into the position of the V_(L).4715 genefragment as a SalI-NotI fragment.

pGOSA.A

This plasmid is derived from both the Fv.4715-myc construct (SEQ ID NO.25) and the scFv.4715-myc construct (SEQ IN NO. 26). An SfiI restrictionsite was introduced between the DNA sequence encoding the (Gly₄Ser)₃linker and the gene fragment encoding the V_(L) of the scFv.4715-mycconstruct (see FIG. 17). This was achieved by replacing the BstEII-SacIfragment of the latter construct by the fragment PCR-I BstEII/SacI (FIG.22) that contains an SfiI site between the DNA encoding the (Gly₄Ser)₃linker and the V_(L).4715 gene fragment. The introduction of the SfiIsite also introduced 4 additional amino acids (AlaGlySerAla) between the(Gly₄Ser)₃ linker and V_(L).4715 resulting in a (Gly₄Ser)₃AlaGlySerAlalinker (linkerA). The oligonucleotides used to produce PCR-I (DBL.5 andDBL.7, see Table 1) were designed to match the sequence of theframework-3 region of V_(H).4715 and to prime at the junction of the DNAencoding the (Gly₄Ser)₃ linker and the V_(L).4715 gene fragment,respectively. Thus pGOSA.A can be indicated as:

pelB-V_(H)4715-linkerA-(SfiI)-V_(L)4715-myc.

pGOSA.B

This plasmid is derived from plasmid Fv.3418 (see FIG. 18). TheXhoI-EcoRI fragment of plasmid Fv.3418 comprising the 3′ end of DNAencoding framework-4 of the V_(L) including the stop codon was removedand replaced by the fragment PCR-IV XhoI/EcoRI (FIG. 23). Theoligonucleotides used to produce PCR-IV (DBL.8 and DBL.6, see Table 1)were designed to match the sequence at the junction of the V_(L) and the(Gly₄Ser)₃ linker perfectly (DBL.8), and to be able to prime at thejunction of the (Gly₄Ser)₃ linker and the V_(H) in pUR.4124 (DBL.6).DBL.6 removed the PstI site in the V_(H) (silent mutation) andintroduced a SalI restriction site at the junction of the (Gly₄Ser)₃linker and the V_(H), thereby replacing the last Ser of the linker by aVal residue resulting in a (Gly₄Ser)₂Gly₄Val linker (linkerV). ThuspGOSA.B can be indicated as:

pelB-V_(H)3418+pelB-V_(L)3418-linkerV-(SalI-EcoRI).

pGOSA.C

This plasmid contains DNA encoding V_(H).4715 linked by the(Gly₄Ser)₃AlaGlySerAla linker to V_(H).3418 (see FIG. 19), thus:

pelB-V_(H)4715-linkerA-V_(H)3418.

This construct was obtained by replacing the SfiI-EcoRI fragment frompGOSA.A encoding V_(L).4715 by the fragment PCR-II SfiI/EcoRI containingthe V_(H).3418 gene. The oligonucleotides used to produce PCR-II (DBL.1and DBL.2, see Table 1) hybridize in the framework-1 and framework-4region of the gene encoding V_(H).3418, respectively. DBL.1 was designedto remove the PstI restriction site (silent mutation) and to introducean SfiI restriction site upstream of the V_(H) gene. DBL.2 destroys theBstEII restriction site in the framework-4 region and introduces an NheIrestriction site downstream of the stopcodon.

pGOSA.D

This plasmid contains a dicistronic operon comprising the V_(H).3418gene and DNA encoding V_(L).3418 linked by the (Gly₄Ser)₂Gly₄Val linkerto V_(L).4715 (see FIG. 20), thus:

pelB-V_(H)3418+peIB-V_(L)3418-linkerV-V_(L)4715.

This construct was obtained by digesting plasmid pGOSA.B with SalI-EcoRIand inserting the fragment PCR-V SalI/EcoRI (FIG. 24) containing theV_(L).4715 gene. The oligonucleotides used to obtain PCR-V (DBL.3 andDBL.9, see Table 1) were designed to match the nucleotide sequence ofthe framework-1 and framework-4 regions of the V_(L).4715 gene,respectively. DBL.3 removed the SacI site from the framework-1 region(silent mutation) and introduced a SalI restriction site upstream of theV_(L).4715 gene. DBL.9 destroyed the XhoI restriction site in theframework-4 region of the V_(L).4715 gene (silent mutation) andintroduced a NotI and an EcoRI restriction site downstream of the stopcodon.

pGOSA.E

This plasmid contains a dicistronic operon comprising DNA encodingV_(H).4715 linked by the (Gly₄Ser)₃AlaGlySerAla linker to V_(H).3418plus DNA encoding V_(L).3418 linked by the (Gly₄Ser)₂Gly₄Val linker toV_(L).4715 (see FIG. 21), thus:

pelB-V_(H)4715-linkerA-V_(H)3418+pelB-V_(L)3418-linkerV-V_(L)4715.

Both translational units are preceded by a ribosome binding site and DNAencoding a pelB leader sequence. This plasmid was obtained by athree-point ligation by mixing the vector resulting from pGOSA.D afterremoval of the V_(H)3418-encoding PstI-SacI insert with the PstI-NheIpGOSA.C insert containing V_(H).4715 linked to V_(H).3418 and thePCR-III NheI/SacI fragment (see FIG. 25). The remaining PstI-SacIPGOSA.D vector contains the 5′ end of the framework-1 region ofV_(H).3418 up to the PstI restriction site and V_(L).3418 linked by the(Gly₄Ser)₂Gly₄Val linker to V_(L).4715 starting from the SacIrestriction site in V_(L).3418. The PstI-NheI pGOSA.C insert containsV_(H).4715 linked by the (Gly₄Ser)₃AlaGlySerAla linker to V_(H).3418,starting from the PstI restriction site in the framework-1 region inV_(H).4715. The NheI-SacI PCR-III fragment provides the ribosome bindingsite and DNA encoding the pelB leader sequence for theV_(L).3418-(Gly₄Ser)₂Gly₄Val-V_(L).4715 construct. The oligonucleotidesDBL.10 and PCR.116 (see Table 1) used to generate PCR-III were designedto match the sequence upstream of the ribosome binding site ofV_(L).4715 in Fv.4715 and to introduce an NheI restriction site(DEL.10), and to match the framework-4 region of V_(L).3418 (PCR.116).

27 1745 base pairs nucleic acid single linear DNA (genomic) unknown 1AAGCTTGCAT GGAAATTCTA TTTCAAGGAG ACAGTCATAA TGAAATACCT ATTGCCTACG 60GCAGCCGCTG GATTGTTATT ACTCGCTGCC CAACCAGCGA TGGCCCAGGT GCAGCTGCAG 120GAGTCAGGGG GAGACTTAGT GAAGCCTGGA GGGTCCCTGA CACTCTCCTG TGCAACCTCT 180GGATTCACTT TCAGTAGTTA TGCCTTTTCT TGGGTCCGCC AGACCTCAGA CAAGAGTCTG 240GAGTGGGTCG CAACCATCAG TAGTACTGAT ACTTATACCT ATTATTCAGA CAATGTGAAG 300GGGCGCTTCA CCATCTCCAG AGACAATGGC AAGAACACCC TGTACCTGCA AATGAGCAGT 360CTGAAGTCTG AGGACACAGC CGTGTATTAC TGTGCAAGAC ATGGGTACTA TGGTAAAGGC 420TATTTTGACT ACTGGGGCCA AGGGACCACG GTCACCGTCT CCTCAGGTGG AGGCGGTTCA 480GGCGGAGGTG GCTCTGGCGG TGGCGGATCG GCCGGTTCGG CCCAGGTCCA GCTGCAACAG 540TCAGGACCTG AGCTGGTAAA GCCTGGGGCT TCAGTGAAGA TGTCCTGCAA GGCTTCTGGA 600TACACATTCA CTAGCTATGT TATGCACTGG GTGAAACAGA AGCCTGGGCA GGGCCTTGAG 660TGGATTGGAT ATATTTATCC TTACAATGAT GGTACTAAGT ACAATGAGAA GTTCAAAGGC 720AAGGCCACAC TGACTTCAGA CAAATCCTCC AGCACAGCCT ACATGGAGCT CAGCAGCCTG 780ACCTCTGAGG ACTCTGCGGT CTATTACTGT TCAAGACGCT TTGACTACTG GGGCCAAGGG 840ACCACCGTCA CCGTCTCCTC ATAATAAGCT AGCGGAGCTG CATGCAAATT CTATTTCAAG 900GAGACAGTCA TAATGAAATA CCTATTGCCT ACGGCAGCCG CTGGATTGTT ATTACTCGCT 960GCCCAACCAG CGATGGCCGA CATCGAGCTC ACCCAGTCTC CATCTTCCAT GTATGCATCT 1020CTAGGAGAGA GAATCACTAT CACTTGCAAG GCGAGTCAGG ACATTAATAC CTATTTAACC 1080TGGTTCCAGC AGAAACCAGG GAAATCTCCC AAGACCCTGA TCTATCGTGC AAACAGATTG 1140CTAGATGGGG TCCCATCAAG GTTCAGTGGC AGTGGATCTG GGCAAGATTA TTCTCTCACC 1200ATCAGCAGCC TGGACTATGA AGATATGGGA ATTTATTATT GTCTACAATA TGATGAGTTG 1260TACACGTTCG GAGGGGGGAC CAAGCTCGAG ATCAAACGGG GTGGAGGCGG TTCAGGCGGA 1320GGTGGCTCTG GCGGTGGCGG AGTCGACATC GAACTCACTC AGTCTCCATT CTCCCTGACT 1380GTGACAGCAG GAGAGAAGGT CACTATGAAT TGCAAGTCCG GTCAGAGTCT GTTAAACAGT 1440GTAAATCAGA GGAACTACTT GACCTGGTAC CAGCAGAAGC CAGGGCAGCC TCCTAAACTG 1500TTGATCTACT GGGCATCCAC TAGGGAATCT GGAGTCCCTG ATCGCTTCAC AGCCAGTGGA 1560TCTGGAACAG ATTTCACTCT CACCATCAGC AGTGTGCAGG CTGAAGACCT GGCAGTTTAT 1620TACTGTCAGA ATGATTATAC TTATCCGTTC ACGTTCGGAG GGGGGACCAA GCTCGAAATC 1680AAACGGGGAT CCGGTAGCGG GAACTCCGGT AAGGGGTACC TGAAGTAATA AGCGGCCGCG 1740AATTC 1745 894 base pairs nucleic acid single linear DNA (genomic)unknown 2 AAGCTTGCAT GCAAATTCTA TTTCAAGGAG ACAGTCATAA TGAAATACCTATTGCCTACG 60 GCAGCCGCTG GATTGTTATT ACTCGCTGCC CAACCAGCGA TGGCCCAGGTGCAGCTGCAG 120 GAGTCAGGAC CTGGCCTGGT GGCGCCCTCA CAGAGCCTGT CCATCACATGCACCGTCTCA 180 GGGTTCTCAT TAACCGGCTA TGGTGTAAAC TGGGTTCGCC AGCCTCCAGGAAAGGGTCTG 240 GAGTGGCTGG GAATGATTTG GGGTGATGGA AACACAGACT ATAATTCAGCTCTCAAATCC 300 AGACTGAGCA TCAGCAAGGA CAACTCCAAG AGCCAAGTTT TCTTAAAAATGAACAGTCTG 360 CACACTGATG ACACAGCCAG GTACTACTGT GCCAGAGAGA GAGATTATAGGCTTGACTAC 420 TGGGGCGAAG GCACCACGGT CACCGTCTCC TCAGGTGGAG GCGGTTCAGGCGGAGGTGGC 480 TCTGGCGGTG GCGGATCGGA CATCGAGCTC ACCCAGTCTC CAGCCTCCCTTTCTGCGTCT 540 GTGGGAGAAA CTGTCACCAT CACATGTCGA GCAAGTGGGA ATATTCACAATTATTTAGCA 600 TGGTATCAGC AGAAACAGGG AAAATCTCCT CAGCTCCTGG TCTATTATACAACAACCTTA 660 GCAGATGGTG TGCCATCAAG GTTCAGTGGC AGTGGATCAG GAACACAATATTCTCTCAAG 720 ATCAACAGCC TGCAACCTGA AGATTTTGGG AGTTATTACT GTCAACATTTTTGGAGTACT 780 CCTCGGACGT TCGGTGGAGG CACCAAGCTC GAGATCAAAC GGGAACAAAAACTCATCTCA 840 GAAGAGGATC TGAATTAATA AGATCAAACG GTAATAAGGA TCCAGCTCGAATTC 894 930 base pairs nucleic acid single linear DNA (genomic) unknown3 AAGCTTGCAT GCAAATTCTA TTTCAAGGAG ACAGTCATAA TGAAATACCT ATTGCCTACG 60GCAGCCGCTG GATTGTTATT ACTCGCTGCC CAACCGGCCA TGGCCCAGGT GCAGCTGCAG 120GAGTCAGGGG GAGACTTAGT GAAGCCTGGA GGGTCCCTGA CACTCTCCTG TGCAACCTCT 180GGATTCACTT TCAGTAGTTA TGCCTTTTCT TGGGTCCGCC AGACCTCAGA CAAGAGTCTG 240GAGTGGGTCG CAACCATCAG TAGTACTGAT ACTTATACCT ATTATTCAGA CAATGTGAAG 300GGGCGCTTCA CCATCTCCAG AGACAATGGC AAGAACACCC TGTACCTGCA AATGAGCAGT 360CTGAAGTCTG AGGACACAGC CGTGTATTAC TGTGCAAGAC ATGGGTACTA TGGTAAAGGC 420TATTTTGACT ACTGGGGCCA AGGGACCACG GTCACCGTCT CCTCAGGTGG AGGCGGTTCA 480GGCGGAGGTG GCTCTGGCGG TGGCGGATCG GACATCGAGC TCACTCAGTC TCCATTCTCC 540CTGACTGTGA CAGCAGGAGA GAAGGTCACT ATGAATTGCA AGTCCGGTCA GAGTCTGTTA 600AACAGTGTAA ATCAGAGGAA CTACTTGACC TGGTACCAGC AGAAGCCAGG GCAGCCTCCT 660AAACTGTTGA TCTACTGGGC ATCCACTAGG GAATCTGGAG TCCCTGATCG CTTCACAGCC 720AGTGGATCTG GAACAGATTT CACTCTCACC ATCAGCAGTG TGCAGGCTGA AGACCTGGCA 780GTTTATTACT GTCAGAATGA TTATACTTAT CCGTTCACGT TCGGAGGGGG GACCAAGCTC 840GAGATCAAAC GGGGATCCGG TAGCGGGAAC TCCGGTAAGG GGTACCTGAA GTAATAAGAT 900CAAACGGTAA TAAGGATCCA GCTCGAATTC 930 156 base pairs nucleic acid singlelinear DNA (genomic) unknown 4 CCATGGGATG GAGCTGTATC ATCCTCTTCTTGGTAGCAAC AGCTACAGGT AAGGGGCTCA 60 CAGTAGCAGG CTTGAGGTCT GGACATATATATGGGTGACA ATGACATCCA CTTTGCCTTT 120 CTCTCCACAG GTGTCCACTC CCAGGTCCAACTGCAG 156 22 base pairs nucleic acid single linear DNA (genomic)unknown 5 AGGTSMAMCT GCAGSAGTCW GG 22 32 base pairs nucleic acid singlelinear DNA (genomic) unknown 6 TGAGGAGACG GTGACCGTGG TCCCTTGGCC CC 32 24base pairs nucleic acid single linear DNA (genomic) unknown 7 GACATTGAGCTCACCCAGTC TCCA 24 22 base pairs nucleic acid single linear DNA(genomic) unknown 8 GTTAGATCTC GAGCTTGGTC CC 22 45 base pairs nucleicacid single linear DNA (genomic) unknown 9 CAGGATCCGG CCGGTTCGGCCCAGGTCCAG CTGCAACAGT CAGGA 45 53 base pairs nucleic acid single linearDNA (genomic) unknown 10 CTACATGAAT TCGCTAGCTT ATTATGAGGA GACGGTGACGGTGGTCCCTT GGC 53 39 base pairs nucleic acid single linear DNA (genomic)unknown 11 ATTGGAGTCG ACATCGAACT CACTCAGTCT CCATTCTCC 39 36 base pairsnucleic acid single linear DNA (genomic) unknown 12 CGAATTCGGATCCCCGTTTG ATTTCGAGCT TGGTCC 36 45 base pairs nucleic acid single linearDNA (genomic) unknown 13 GAGCGCGAGC TCGGCCGAAC CGGCCGATCC GCCACCGCCAGAGCC 45 36 base pairs nucleic acid single linear DNA (genomic) unknown14 ATTGTCGAAT TCGTCGACTC CGCCACCGCC AGAGCC 36 57 base pairs nucleic acidsingle linear DNA (genomic) unknown 15 AGCTTCTAGA CCACCATGGA AAACTGCAGAGCTCAAAAGC TAGCGCGGCG GCTCTAG 57 57 base pairs nucleic acid singlelinear DNA (genomic) unknown 16 AATTCTAGAG CGGCCGCGCT AGCTTTTGAGCTCTGCAGTT TTCCATGGTG GTCTAGA 57 40 base pairs nucleic acid singlelinear DNA (genomic) unknown 17 ACGGGTGAGC TCGATGTCGG AGTGGACACCTGTGGAGAGA 40 24 base pairs nucleic acid single linear DNA (genomic)unknown 18 GGAAACAGCT ATGACCATGA TTAC 24 25 base pairs nucleic acidsingle linear other nucleic acid /desc = “synthetic DNA” unknown primerDBL.7 19 CACCATCTCC AGAGACAATG GCAAG 25 23 base pairs nucleic acidsingle linear other nucleic acid /desc = “synthetic DNA” unknown primerDBL.8 20 ACCAAGCTCG AGATCAAACG GGG 23 50 base pairs nucleic acid singlelinear other nucleic acid /desc = “synthetic DNA” unknown primer DBL.921 TGAAGTGAAT TCGCGGCCGC TTATTACCGT TTGATTTCGA GCTTGGTCCC 50 36 basepairs nucleic acid single linear other nucleic acid /desc = “syntheticDNA” unknown primer DBL.10 22 TAATAAGCTA GCGGAGCTGC ATGCAAATTC TATTTC 36737 base pairs nucleic acid double linear other nucleic acid /desc =“cDNA domains with synthetic linker(s)” unknown EcoRI-HindIII insert ofpUR4124 CDS 11..730 /product= “VLlys-GS-VHlys” mat_peptide 11..334/product= “VLlys” misc_RNA 335..379 /product= “(Gly4Ser)3 linker”mat_peptide 380..727 /product= “VHlys” 23 GAATTCGGCC GAC ATC GAG CTC ACCCAG TCT CCA GCC TCC CTT TCT GCG 49 Asp Ile Glu Leu Thr Gln Ser Pro AlaSer Leu Ser Ala 1 5 10 TCT GTG GGA GAA ACT GTC ACC ATC ACA TGT CGA GCAAGT GGG AAT ATT 97 Ser Val Gly Glu Thr Val Thr Ile Thr Cys Arg Ala SerGly Asn Ile 15 20 25 CAC AAT TAT TTA GCA TGG TAT CAG CAG AAA CAG GGA AAATCT CCT CAG 145 His Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys SerPro Gln 30 35 40 45 CTC CTG GTC TAT TAT ACA ACA ACC TTA GCA GAT GGT GTGCCA TCA AGG 193 Leu Leu Val Tyr Tyr Thr Thr Thr Leu Ala Asp Gly Val ProSer Arg 50 55 60 TTC AGT GGC AGT GGA TCA GGA ACA CAA TAT TCT CTC AAG ATCAAC AGC 241 Phe Ser Gly Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile AsnSer 65 70 75 CTG CAA CCT GAA GAT TTT GGG AGT TAT TAC TGT CAA CAT TTT TGGAGT 289 Leu Gln Pro Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His Phe Trp Ser80 85 90 ACT CCT CGG ACG TTC GGT GGA GGG ACC AAG CTC GAG ATC AAA CGG GGT337 Thr Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Gly 95100 105 GGA GGC GGT TCA GGC GGA GGT GGC TCT GGC GGT GGC GGA TCG CAG GTG385 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val 110115 120 125 CAG CTG CAG GAG TCA GGA CCT GGC CTG GTG GCG CCC TCA CAG AGCCTG 433 Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu130 135 140 TCC ATC ACA TGC ACC GTC TCA GGG TTC TCA TTA ACC GGC TAT GGTGTA 481 Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Gly Tyr Gly Val145 150 155 AAC TGG GTT CGC CAG CCT CCA GGA AAG GGT CTG GAG TGG CTG GGAATG 529 Asn Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu Gly Met160 165 170 ATT TGG GGT GAT GGA AAC ACA GAC TAT AAT TCA GCT CTC AAA TCCAGA 577 Ile Trp Gly Asp Gly Asn Thr Asp Tyr Asn Ser Ala Leu Lys Ser Arg175 180 185 CTG AGC ATC AGC AAG GAC AAC TCC AAG AGC CAA GTT TTC TTA AAAATG 625 Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met190 195 200 205 AAC AGT CTG CAC ACT GAT GAC ACA GCC AGG TAC TAC TGT GCCAGA GAG 673 Asn Ser Leu His Thr Asp Asp Thr Ala Arg Tyr Tyr Cys Ala ArgGlu 210 215 220 AGA GAT TAT AGG CTT GAC TAC TGG GGC CAA GGG ACC ACG GTCACC GTC 721 Arg Asp Tyr Arg Leu Asp Tyr Trp Gly Gln Gly Thr Thr Val ThrVal 225 230 235 TCC TCA TGA TAAGCTT 737 Ser Ser * 240 920 base pairsnucleic acid double linear other nucleic acid /desc = “cDNA domains withsynthetic linker(s)” unknown HindIII-EcoRI insert Fv.3418 CDS 36..443/product= “pelB-VH3418” sig_peptide 36..101 /product= “pectate lyase”mat_peptide 102..440 /product= “VH3418” CDS 495..884 /product=“pelB-VL4318” sig_peptide 495..560 /product= “pectate lyase” mat_peptide561..881 /product= “VL3418” 24 AAGCTTGCAA ATTCTATTTC AAGGAGACAG TCATAATG AAA TAC CTA TTG CCT 53 Met Lys Tyr Leu Leu Pro -22 -20 ACG GCA GCCGCT GGA TTG TTA TTA CTC GCT GCC CAA CCA GCG ATG GCC 101 Thr Ala Ala AlaGly Leu Leu Leu Leu Ala Ala Gln Pro Ala Met Ala -15 -10 -5 CAG GTG CAGCTG CAG CAG TCA GGA CCT GAG CTG GTA AAG CCT GGG GCT 149 Gln Val Gln LeuGln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 TCA GTG AAGATG TCC TGC AAG GCT TCT GGA TAC ACA TTC ACT AGC TAT 197 Ser Val Lys MetSer Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 GTT ATG CAC TGGGTG AAA CAG AAG CCT GGG CAG GGC CTT GAG TGG ATT 245 Val Met His Trp ValLys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 GGA TAT ATT TAT CCTTAC AAT GAT GGT ACT AAG TAC AAT GAG AAG TTC 293 Gly Tyr Ile Tyr Pro TyrAsn Asp Gly Thr Lys Tyr Asn Glu Lys Phe 50 55 60 AAA GGC AAG GCC ACA CTGACT TCA GAC AAA TCC TCC AGC ACA GCC TAC 341 Lys Gly Lys Ala Thr Leu ThrSer Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 ATG GAG CTC AGC AGC CTGACC TCT GAG GAC TCT GCG GTC TAT TAC TGT 389 Met Glu Leu Ser Ser Leu ThrSer Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 TCA AGA CGC TTT GAC TAC TGGGGC CAA GGG ACC ACG GTC ACC GTC TCC 437 Ser Arg Arg Phe Asp Tyr Trp GlyGln Gly Thr Thr Val Thr Val Ser 100 105 110 TCA TAA TAAGAGCTATGGGAGCTTGC ATGCAAATTC TATTTCAAGG AGACAGTCAT 493 Ser * A ATG AAA TAC CTATTG CCT ACG GCA GCC GCT GGA TTG TTA TTA CTC 539 Met Lys Tyr Leu Leu ProThr Ala Ala Ala Gly Leu Leu Leu Leu -22 -20 -15 -10 GCT GCC CAA CCA GCGATG GCC GAC ATC GAG CTC ACC CAG TCT CCA TCT 587 Ala Ala Gln Pro Ala MetAla Asp Ile Glu Leu Thr Gln Ser Pro Ser -5 1 5 TCC ATG TAT GCA TCT CTAGGA GAG AGA ATC ACT ATC ACT TGC AAG GCG 635 Ser Met Tyr Ala Ser Leu GlyGlu Arg Ile Thr Ile Thr Cys Lys Ala 10 15 20 25 AGT CAG GAC ATT AAT ACCTAT TTA ACC TGG TTC CAG CAG AAA CCA GGG 683 Ser Gln Asp Ile Asn Thr TyrLeu Thr Trp Phe Gln Gln Lys Pro Gly 30 35 40 AAA TCT CCC AAG ACC CTG ATCTAT CGT GCA AAC AGA TTG CTA GAT GGG 731 Lys Ser Pro Lys Thr Leu Ile TyrArg Ala Asn Arg Leu Leu Asp Gly 45 50 55 GTC CCA TCA AGG TTC AGT GGC AGTGGA TCT GGG CAA GAT TAT TCT CTC 779 Val Pro Ser Arg Phe Ser Gly Ser GlySer Gly Gln Asp Tyr Ser Leu 60 65 70 ACC ATC AGC AGC CTG GAC TAT GAA GATATG GGA ATT TAT TAT TGT CTA 827 Thr Ile Ser Ser Leu Asp Tyr Glu Asp MetGly Ile Tyr Tyr Cys Leu 75 80 85 CAA TAT GAT GAG TTG TAC ACG TTC GGA GGGGGG ACC AAG CTC GAG ATC 875 Gln Tyr Asp Glu Leu Tyr Thr Phe Gly Gly GlyThr Lys Leu Glu Ile 90 95 100 105 AAA CGG TAA TAATGATCAA ACGGTATAAGGATCCAGCTC GAATTC 920 Lys Arg * 999 base pairs nucleic acid doublelinear other nucleic acid /desc = “cDNA domains with syntheticlinker(s)” unknown HindIII-EcoRI insert of Fv.4715-myc CDS 40..468/product= “pelB-VH4715” sig_peptide 40..105 /product= “pectate lyase”mat_peptide 106..465 /product= “VH4715” CDS 520..963 /product=“pelB-VL4715-myc” sig_peptide 520..585 /product= “pectate lyase”mat_peptide 586..927 /product= “VL4715” misc_RNA 928..960 /product=“myc-tag” 25 AAGCTTGCAT GCAAATTCTA TTTCAAGGAG ACAGTCATA ATG AAA TAC CTATTG 54 Met Lys Tyr Leu Leu -22 -20 CCT ACG GCA GCC GCT GGA TTG TTA TTACTC GCT GCC CAA CCA GCG ATG 102 Pro Thr Ala Ala Ala Gly Leu Leu Leu LeuAla Ala Gln Pro Ala Met -15 -10 -5 GCC CAG GTG CAG CTG CAG GAG TCA GGGGGA GAC TTA GTG AAG CCT GGA 150 Ala Gln Val Gln Leu Gln Glu Ser Gly GlyAsp Leu Val Lys Pro Gly 1 5 10 15 GGG TCC CTG ACA CTC TCC TGT GCA ACCTCT GGA TTC ACT TTC AGT AGT 198 Gly Ser Leu Thr Leu Ser Cys Ala Thr SerGly Phe Thr Phe Ser Ser 20 25 30 TAT GCC TTT TCT TGG GTC CGC CAG ACC TCAGAC AAG AGT CTG GAG TGG 246 Tyr Ala Phe Ser Trp Val Arg Gln Thr Ser AspLys Ser Leu Glu Trp 35 40 45 GTC GCA ACC ATC AGT AGT ACT GAT ACT TAT ACCTAT TAT TCA GAC AAT 294 Val Ala Thr Ile Ser Ser Thr Asp Thr Tyr Thr TyrTyr Ser Asp Asn 50 55 60 GTG AAG GGG CGC TTC ACC ATC TCC AGA GAC AAT GGCAAG AAC ACC CTG 342 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly LysAsn Thr Leu 65 70 75 TAC CTG CAA ATG AGC AGT CTG AAG TCT GAG GAC ACA GCCGTG TAT TAC 390 Tyr Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala ValTyr Tyr 80 85 90 95 TGT GCA AGA CAT GGG TAC TAT GGT AAA GGC TAT TTT GACTAC TGG GGC 438 Cys Ala Arg His Gly Tyr Tyr Gly Lys Gly Tyr Phe Asp TyrTrp Gly 100 105 110 CAA GGG ACC ACG GTC ACC GTC TCC TCA TAA TAAGAGCTATGGGAGCTTGC 488 Gln Gly Thr Thr Val Thr Val Ser Ser * 115 120 ATGCAAATTCTATTTCAAGG AGACAGTCAT A ATG AAA TAC CTA TTG CCT ACG 540 Met Lys Tyr LeuLeu Pro Thr -22 -20 GCA GCC GCT GGA TTG TTA TTA CTC GCT GCC CAA CCA GCGATG GCC GAC 588 Ala Ala Ala Gly Leu Leu Leu Leu Ala Ala Gln Pro Ala MetAla Asp -15 -10 -5 1 ATC GAG CTC ACT CAG TCT CCA TTC TCC CTG ACT GTG ACAGCA GGA GAG 636 Ile Glu Leu Thr Gln Ser Pro Phe Ser Leu Thr Val Thr AlaGly Glu 5 10 15 AAG GTC ACT ATG AAT TGC AAG TCC GGT CAG AGT CTG TTA AACAGT GTA 684 Lys Val Thr Met Asn Cys Lys Ser Gly Gln Ser Leu Leu Asn SerVal 20 25 30 AAT CAG AGG AAC TAC TTG ACC TGG TAC CAG CAG AAG CCA GGG CAGCCT 732 Asn Gln Arg Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln Pro35 40 45 CCT AAA CTG TTG ATC TAC TGG GCA TCC ACT AGG GAA TCT GGA GTC CCT780 Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro 5055 60 65 GAT CGC TTC ACA GCC AGT GGA TCT GGA ACA GAT TTC ACT CTC ACC ATC828 Asp Arg Phe Thr Ala Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 7075 80 AGC AGT GTG CAG GCT GAA GAC CTG GCA GTT TAT TAC TGT CAG AAT GAT876 Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn Asp 8590 95 TAT ACT TAT CCG TTC ACG TTC GGA GGG GGG ACC AAG CTC GAG ATC AAA924 Tyr Thr Tyr Pro Phe Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100105 110 CGG GAA CAA AAA CTC ATC TCA GAA GAG GAT CTG AAT TAA TAAGATCAAA973 Arg Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn * 115 120 125CGGTAATAAG GATCCAGCTC GAATTC 999 924 base pairs nucleic acid doublelinear other nucleic acid /desc = “cDNA domains with syntheticlinker(s)” unknown HindIII-EcoRI insert of scFv.4715-myc sig_peptide40..105 /product= “pectate lyase” mat_peptide 106..465 /product=“VH4715” misc_RNA 466..510 /product= “(Gly4Ser)3-linker” mat_peptide511..852 /product= “VL4715” misc_RNA 853..885 /product= “myc-tag” CDS40..888 /product= “pelB-VH4715-(Gly4Ser)3-VL4715-myc” 26 AAGCTTGCATGCAAATTCTA TTTCAAGGAG ACAGTCATA ATG AAA TAC CTA TTG 54 Met Lys Tyr LeuLeu -22 -20 CCT ACG GCA GCC GCT GGA TTG TTA TTA CTC GCT GCC CAA CCA GCGATG 102 Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala Ala Gln Pro Ala Met-15 -10 -5 GCC CAG GTG CAG CTG CAG GAG TCA GGG GGA GAC TTA GTG AAG CCTGGA 150 Ala Gln Val Gln Leu Gln Glu Ser Gly Gly Asp Leu Val Lys Pro Gly1 5 10 15 GGG TCC CTG ACA CTC TCC TGT GCA ACC TCT GGA TTC ACT TTC AGTAGT 198 Gly Ser Leu Thr Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Ser Ser20 25 30 TAT GCC TTT TCT TGG GTC CGC CAG ACC TCA GAC AAG AGT CTG GAG TGG246 Tyr Ala Phe Ser Trp Val Arg Gln Thr Ser Asp Lys Ser Leu Glu Trp 3540 45 GTC GCA ACC ATC AGT AGT ACT GAT ACT TAT ACC TAT TAT TCA GAC AAT294 Val Ala Thr Ile Ser Ser Thr Asp Thr Tyr Thr Tyr Tyr Ser Asp Asn 5055 60 GTG AAG GGG CGC TTC ACC ATC TCC AGA GAC AAT GGC AAG AAC ACC CTG342 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Asn Thr Leu 6570 75 TAC CTG CAA ATG AGC AGT CTG AAG TCT GAG GAC ACA GCC GTG TAT TAC390 Tyr Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr 8085 90 95 TGT GCA AGA CAT GGG TAC TAT GGT AAA GGC TAT TTT GAC TAC TGG GGC438 Cys Ala Arg His Gly Tyr Tyr Gly Lys Gly Tyr Phe Asp Tyr Trp Gly 100105 110 CAA GGG ACC ACG GTC ACC GTC TCC TCA GGT GGA GGC GGT TCA GGC GGA486 Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly 115120 125 GGT GGC TCT GGC GGT GGC GGA TCG GAC ATC GAG CTC ACT CAG TCT CCA534 Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Glu Leu Thr Gln Ser Pro 130135 140 TTC TCC CTG ACT GTG ACA GCA GGA GAG AAG GTC ACT ATG AAT TGC AAG582 Phe Ser Leu Thr Val Thr Ala Gly Glu Lys Val Thr Met Asn Cys Lys 145150 155 TCC GGT CAG AGT CTG TTA AAC AGT GTA AAT CAG AGG AAC TAC TTG ACC630 Ser Gly Gln Ser Leu Leu Asn Ser Val Asn Gln Arg Asn Tyr Leu Thr 160165 170 175 TGG TAC CAG CAG AAG CCA GGG CAG CCT CCT AAA CTG TTG ATC TACTGG 678 Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp180 185 190 GCA TCC ACT AGG GAA TCT GGA GTC CCT GAT CGC TTC ACA GCC AGTGGA 726 Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Ala Ser Gly195 200 205 TCT GGA ACA GAT TTC ACT CTC ACC ATC AGC AGT GTG CAG GCT GAAGAC 774 Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp210 215 220 CTG GCA GTT TAT TAC TGT CAG AAT GAT TAT ACT TAT CCG TTC ACGTTC 822 Leu Ala Val Tyr Tyr Cys Gln Asn Asp Tyr Thr Tyr Pro Phe Thr Phe225 230 235 GGA GGG GGG ACC AAG CTC GAG ATC AAA CGG GAA CAA AAA CTC ATCTCA 870 Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Glu Gln Lys Leu Ile Ser240 245 250 255 GAA GAG GAT CTG AAT TAA TAAGATCAAA CGGTAATAAG GATCCAGCTCGAATTC 924 Glu Glu Asp Leu Asn * 260 1706 base pairs nucleic acid doublelinear other nucleic acid /desc = “cDNA domains with syntheticlinker(s)” unknown HindIII-EcoRI insert of pGOSA.E CDS 40..864 /product=“pelB-VH4715-LiA-VH3418” sig_peptide 40..105 /product= “pectate lyase”mat_peptide 106..465 /product= “VH4715” misc_RNA 466..522 /product=“linkerA (Gly4Ser)3AlaGlySerAla” mat_peptide 523..861 /product= “VH3418”CDS 913..1689 /product= “pelB-VL3418-LiV-VL4715” sig_peptide 913..978/product= “pectate lyase” mat_peptide 979..1299 /product= “VL3418”misc_RNA 1300..1344 /product= “linker V (Gly4Ser)2Gly4Val” mat_peptide1345..1686 /product= “VL4715” 27 AAGCTTGCAT GGAAATTCTA TTTCAAGGAGACAGTCATA ATG AAA TAC CTA TTG 54 Met Lys Tyr Leu Leu -22 -20 CCT ACG GCAGCC GCT GGA TTG TTA TTA CTC GCT GCC CAA CCA GCG ATG 102 Pro Thr Ala AlaAla Gly Leu Leu Leu Leu Ala Ala Gln Pro Ala Met -15 -10 -5 GCC CAG GTGCAG CTG CAG GAG TCA GGG GGA GAC TTA GTG AAG CCT GGA 150 Ala Gln Val GlnLeu Gln Glu Ser Gly Gly Asp Leu Val Lys Pro Gly 1 5 10 15 GGG TCC CTGACA CTC TCC TGT GCA ACC TCT GGA TTC ACT TTC AGT AGT 198 Gly Ser Leu ThrLeu Ser Cys Ala Thr Ser Gly Phe Thr Phe Ser Ser 20 25 30 TAT GCC TTT TCTTGG GTC CGC CAG ACC TCA GAC AAG AGT CTG GAG TGG 246 Tyr Ala Phe Ser TrpVal Arg Gln Thr Ser Asp Lys Ser Leu Glu Trp 35 40 45 GTC GCA ACC ATC AGTAGT ACT GAT ACT TAT ACC TAT TAT TCA GAC AAT 294 Val Ala Thr Ile Ser SerThr Asp Thr Tyr Thr Tyr Tyr Ser Asp Asn 50 55 60 GTG AAG GGG CGC TTC ACCATC TCC AGA GAC AAT GGC AAG AAC ACC CTG 342 Val Lys Gly Arg Phe Thr IleSer Arg Asp Asn Gly Lys Asn Thr Leu 65 70 75 TAC CTG CAA ATG AGC AGT CTGAAG TCT GAG GAC ACA GCC GTG TAT TAC 390 Tyr Leu Gln Met Ser Ser Leu LysSer Glu Asp Thr Ala Val Tyr Tyr 80 85 90 95 TGT GCA AGA CAT GGG TAC TATGGT AAA GGC TAT TTT GAC TAC TGG GGC 438 Cys Ala Arg His Gly Tyr Tyr GlyLys Gly Tyr Phe Asp Tyr Trp Gly 100 105 110 CAA GGG ACC ACG GTC ACC GTCTCC TCA GGT GGA GGC GGT TCA GGC GGA 486 Gln Gly Thr Thr Val Thr Val SerSer Gly Gly Gly Gly Ser Gly Gly 115 120 125 GGT GGC TCT GGC GGT GGC GGATCG GCC GGT TCG GCC CAG GTC CAG CTG 534 Gly Gly Ser Gly Gly Gly Gly SerAla Gly Ser Ala Gln Val Gln Leu 130 135 140 CAA CAG TCA GGA CCT GAG CTGGTA AAG CCT GGG GCT TCA GTG AAG ATG 582 Gln Gln Ser Gly Pro Glu Leu ValLys Pro Gly Ala Ser Val Lys Met 145 150 155 TCC TGC AAG GCT TCT GGA TACACA TTC ACT AGC TAT GTT ATG CAC TGG 630 Ser Cys Lys Ala Ser Gly Tyr ThrPhe Thr Ser Tyr Val Met His Trp 160 165 170 175 GTG AAA CAG AAG CCT GGGCAG GGC CTT GAG TGG ATT GGA TAT ATT TAT 678 Val Lys Gln Lys Pro Gly GlnGly Leu Glu Trp Ile Gly Tyr Ile Tyr 180 185 190 CCT TAC AAT GAT GGT ACTAAG TAC AAT GAG AAG TTC AAA GGC AAG GCC 726 Pro Tyr Asn Asp Gly Thr LysTyr Asn Glu Lys Phe Lys Gly Lys Ala 195 200 205 ACA CTG ACT TCA GAC AAATCC TCC AGC ACA GCC TAC ATG GAG CTC AGC 774 Thr Leu Thr Ser Asp Lys SerSer Ser Thr Ala Tyr Met Glu Leu Ser 210 215 220 AGC CTG ACC TCT GAG GACTCT GCG GTC TAT TAC TGT TCA AGA CGC TTT 822 Ser Leu Thr Ser Glu Asp SerAla Val Tyr Tyr Cys Ser Arg Arg Phe 225 230 235 GAC TAC TGG GGC CAA GGGACC ACC GTC ACC GTC TCC TCA TAA 864 Asp Tyr Trp Gly Gln Gly Thr Thr ValThr Val Ser Ser * 240 245 250 TAAGCTAGCG GAGCTGCATG CAAATTCTATTTCAAGGAGA CAGTCATA ATG AAA TAC 921 Met Lys Tyr -22 -20 CTA TTG CCT ACGGCA GCC GCT GGA TTG TTA TTA CTC GCT GCC CAA CCA 969 Leu Leu Pro Thr AlaAla Ala Gly Leu Leu Leu Leu Ala Ala Gln Pro -15 -10 -5 GCG ATG GCC GACATC GAG CTC ACC CAG TCT CCA TCT TCC ATG TAT GCA 1017 Ala Met Ala Asp IleGlu Leu Thr Gln Ser Pro Ser Ser Met Tyr Ala 1 5 10 TCT CTA GGA GAG AGAATC ACT ATC ACT TGC AAG GCG AGT CAG GAC ATT 1065 Ser Leu Gly Glu Arg IleThr Ile Thr Cys Lys Ala Ser Gln Asp Ile 15 20 25 AAT ACC TAT TTA ACC TGGTTC CAG CAG AAA CCA GGG AAA TCT CCC AAG 1113 Asn Thr Tyr Leu Thr Trp PheGln Gln Lys Pro Gly Lys Ser Pro Lys 30 35 40 45 ACC CTG ATC TAT CGT GCAAAC AGA TTG CTA GAT GGG GTC CCA TCA AGG 1161 Thr Leu Ile Tyr Arg Ala AsnArg Leu Leu Asp Gly Val Pro Ser Arg 50 55 60 TTC AGT GGC AGT GGA TCT GGGCAA GAT TAT TCT CTC ACC ATC AGC AGC 1209 Phe Ser Gly Ser Gly Ser Gly GlnAsp Tyr Ser Leu Thr Ile Ser Ser 65 70 75 CTG GAC TAT GAA GAT ATG GGA ATTTAT TAT TGT CTA CAA TAT GAT GAG 1257 Leu Asp Tyr Glu Asp Met Gly Ile TyrTyr Cys Leu Gln Tyr Asp Glu 80 85 90 TTG TAC ACG TTC GGA GGG GGG ACC AAGCTC GAG ATC AAA CGG GGT GGA 1305 Leu Tyr Thr Phe Gly Gly Gly Thr Lys LeuGlu Ile Lys Arg Gly Gly 95 100 105 GGC GGT TCA GGC GGA GGT GGC TCT GGCGGT GGC GGA GTC GAC ATC GAA 1353 Gly Gly Ser Gly Gly Gly Gly Ser Gly GlyGly Gly Val Asp Ile Glu 110 115 120 125 CTC ACT CAG TCT CCA TTC TCC CTGACT GTG ACA GCA GGA GAG AAG GTC 1401 Leu Thr Gln Ser Pro Phe Ser Leu ThrVal Thr Ala Gly Glu Lys Val 130 135 140 ACT ATG AAT TGC AAG TCC GGT CAGAGT CTG TTA AAC AGT GTA AAT CAG 1449 Thr Met Asn Cys Lys Ser Gly Gln SerLeu Leu Asn Ser Val Asn Gln 145 150 155 AGG AAC TAC TTG ACC TGG TAC CAGCAG AAG CCA GGG CAG CCT CCT AAA 1497 Arg Asn Tyr Leu Thr Trp Tyr Gln GlnLys Pro Gly Gln Pro Pro Lys 160 165 170 CTG TTG ATC TAC TGG GCA TCC ACTAGG GAA TCT GGA GTC CCT GAT CGC 1545 Leu Leu Ile Tyr Trp Ala Ser Thr ArgGlu Ser Gly Val Pro Asp Arg 175 180 185 TTC ACA GCC AGT GGA TCT GGA ACAGAT TTC ACT CTC ACC ATC AGC AGT 1593 Phe Thr Ala Ser Gly Ser Gly Thr AspPhe Thr Leu Thr Ile Ser Ser 190 195 200 205 GTG CAG GCT GAA GAC CTG GCAGTT TAT TAC TGT CAG AAT GAT TAT ACT 1641 Val Gln Ala Glu Asp Leu Ala ValTyr Tyr Cys Gln Asn Asp Tyr Thr 210 215 220 TAT CCG TTC ACG TTC GGA GGGGGG ACC AAG CTC GAA ATC AAA CGG TAA 1689 Tyr Pro Phe Thr Phe Gly Gly GlyThr Lys Leu Glu Ile Lys Arg * 225 230 235 TAAGCGGCCG CGAATTC 1706

What is claimed is:
 1. A multivalent antigen binding protein comprising:a first polypeptide comprising, in series, three or more variabledomains of an antibody heavy chain; and a second polypeptide comprising,in series, three or more variable domains of an antibody light chain,said first and second polypeptides being linked by association of therespective heavy chain and light chain variable domains, each associatedvariable domain pair forming an antigen binding site.
 2. A proteinaccording to claim 1 comprising a trivalent antigen binding protein. 3.A protein according to claim 1 or claim 2 wherein the variable domainsof the antibody heavy chain of said first polypeptide are linked by apeptide linker and the variable domains of the antibody light chain ofsaid second polypeptide are linked by a peptide linker.
 4. A proteinaccording to any one of claims 1 to 3 wherein the associated variabledomain pair binding sites are able to bind different epitopes from eachother.
 5. A protein according to any one of claims 1 to 3 wherein theassociated variable domain pair binding sites are able to bind the sameepitope as each other.
 6. A process for preparing a multivalent antigenbinding protein according to any one of claims 1 to 5 comprising (i)transforming one or more hosts by incorporating genes encoding saidfirst and second polypeptides; (ii) expressing said genes and said hostor hosts; and (iii) allowing said first and second polypeptides toassociate to form the protein.
 7. A protein according to any one ofclaims 1 to 5 for use in medicine.
 8. A diagnostic or therapeuticcomposition comprising a protein according to any one of claims 1 to 5.9. A method of diagnosis or therapy comprising administering a proteinaccording to any one of claims 1 to
 5. 10. A method of preparing anagent for diagnosis or therapy comprising combining a compositionaccording to claim 8 with at least one suitable pharmaceutical carrieror excipient.
 11. A method for immunoassay or purification comprisingcontacting a composition according to one of claims 1 to 5 with a testsample.